On the properties of Calcein Blue

A satisfactory method for the preparation of Calcein Blue has been devised. Elemental analysis, equivalent weight by neutralization, and the NMR spectrum show the compound to be 4-methylumbelliferone-8-methyleneiminodiacetic acid·0·25H₂O. The ultraviolet absorbance and fluorescence have been studied as a function of pH and, combined with potentiometric titration and solubility date, have yielded for the acid dissociation constants the values pK₁ = 3·0, pK₂ = 6·9, and pK₃ = 11·3. These acid functions are identified respectively as carboxyl, phenol, and ammonium ion, the free Calcein Blue being a zwitter-ion. Calcein Blue fluoresces in both acidic and basic solution when excited at a suitable wavelength. The fluorescence of the doubly-charged anion formed on the neutralization of the phenol group, when excited at 360 nm, reaches a maximum at pH 9, and decreases to zero with the neutralization of the ammonium ion; the wavelength of maximum emission is 455 nm. In the presence of calcium, the fluorescence increases with alkalinity up to pH 9 and then remains constant. The calcium derivative is a 1:1 compound, formation constant 10^7·1. The fluorescence of Calcein Blue at all pH values is quenched by copper(II). The calcium derivative is changed on standing in highly alkaline solution, presumably by ring opening, to another fluorescent material; thus Calcein Blue, although satisfactory as an indicator, is not useful for the direct fluorometric determination of calcium.